A novel enzyme-modified cheddar cheese was prepared and the molecular mechanism of cheese flavor compensation by synergistic action of cell-free extracts and enzyme systems was investigated. By comparing five different protease-peptidase combinations, the group of neutral protease and flavor protease was found to increase the total leucine, valine, and isoleucine content (17.056 ± 0.136 g/kg) and the soluble nitrogen content was up to the level of a 12-month-matured cheese. Molecular docking and molecular dynamics simulations demonstrated their mode of action on four monomeric caseins. Adding a cell-free extract resulted in volatile flavor substances in the enzyme-modified cheese that were closest to those in the 12-month-matured cheese. This might be due to the flavor compensation effect of the conversion of leucine to 3-methylbutyraldehyde by transaminases and decarboxylases, and the conversion of 3-methylbutyric acid to 3-methylbutyraldehyde by ketoacid dehydrogenase and aldehyde dehydrogenase. This is essential for the enzyme modified cheddar cheese preparation.
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